Direct and simultaneous quantitation of 5-aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry

Cancer Studies and Molecular Medicine, RKCSB, University of Leicester, Leicester, LE2 7LX, UK.
Biomedical Chromatography (Impact Factor: 1.72). 02/2013; 27(2). DOI: 10.1002/bmc.2843
Source: PubMed


Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias. Copyright © 2012 John Wiley & Sons, Ltd.

Download full-text


Available from: David C Rees, Jan 26, 2015
1 Follower
93 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background. 1H-NMR is a non-biased technique for the quantification of small molecules that could result in the identification and characterization of potential biomarkers with prognostic value and contribute to better understand pathophysiology of diseases. Methods. In this study, we used 1H-NMR spectroscopy to analyze the urinary metabolome of patients with Acute Intermittent Porphyria (AIP), an inherited metabolic disorder of heme biosynthesis in which an accumulation of the heme precursors 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) promotes sudden neuro-visceral attacks which can be life-threatening. Our objectives were (1) to demonstrate the usefulness of 1H-NMR to identify and quantify ALA and PBG in urines from AIP patients, and (2) to identify metabolites that would predict the response to AIP crisis treatment and reflect differential metabolic reprogramming. Results. Our results indicate that 1H-NMR can help to diagnose AIP attacks based on the identification of ALA and PBG. We also show that glycin concentration increases in urines from patients with frequent recurrences at the end of the treatment, after an initial decrease, whereas PBG concentration remains low. Although the reasons for this altered are elusive, these findings indicate that a glycin metabolic reprogramming occurs in AIPr patients and is associated with recurrence Conclusion. Our results validate the proof of concept of the usefulness of 1H-NMR spectroscopy in clinical chemistry for the diagnosis of acute attack of AIP, and identify urinary glycin as a potential marker of recurrence of AIP acute attacks.
    Analytical Chemistry 01/2014; 86(4). DOI:10.1021/ac403837r · 5.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The retention behavior of several series of free α- and ω-amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post-column derivatization with fluorescence detection in order to describe the retention mechanism of tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms (nC ) in aliphatic chains of the individual homologues of α- and ω-amino acids and the logarithm of the partition coefficient (log D) on retention was also a part of the presented study. A good correlation (r > 0.98) between the log k and log D values of amino acids or nC , respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α-amino acids, ω-amino acids and positional isomers of amino acids was based on the log D values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω-amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α-amino acids remained essentially unchanged on all tested stationary phases. This article is protected by copyright. All rights reserved.
    Journal of Separation Science 04/2014; 37(7). DOI:10.1002/jssc.201301348 · 2.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: There is an increasing need for new analytical methods that can handle a large number of analytes in complex matrices. Hydrophilic interaction chromatography (HILIC) has recently been demonstrated as an important supplement to reversed-phase liquid chromatography for polar analytes, particularly endogenous compounds. With the increasing popularity of HILIC, progressively more polar phases with diverse functional groups have been developed. In addition, the coupling of HILIC to mass spectrometry offers the advantages of improved sensitivity by employing an organic-rich mobile phase. This article reviews recent applications of HILIC for the analysis of endogenous and pharmaceutical compounds in plasma samples. Furthermore, based on recent studies, we provide a discussion of column selection, sample pretreatment for HILIC analysis, and detection sensitivity.
    Bioanalysis 09/2014; 6(18):2421-39. DOI:10.4155/bio.14.173 · 3.00 Impact Factor
Show more